RESEARCH LETTER
Comparison of the effects of silver phosphate and selenium
nanoparticles on Staphylococcus aureus growth reveals
potential for selenium particles to prevent infection
Dagmar Chudobova1, Kristyna Cihalova1, Simona Dostalova1, Branislav Ruttkay-Nedecky1,2,
Miguel Angel Merlos Rodrigo1,2, Katerina Tmejova1,2, Pavel Kopel1,2, Lukas Nejdl1, Jiri Kudr1,
Jaromir Gumulec1,3, Sona Krizkova1,2, Jindrich Kynicky1,2, Rene Kizek1,2 & Vojtech Adam1,2
1
Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic; 2Department of Chemistry and Biochemistry,
Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic; and 3Department of Pathological Physiology, Faculty of Medicine,
Masaryk University, Brno, Czech Republic
Correspondence: Vojtech Adam,
Department of Chemistry and Biochemistry,
Mendel University in Brno, Zemedelska 1,
CZ-613 00 Brno, Czech Republic.
Tel.: +420 545 133 350;
fax: +420 545 212 044;
e-mail: vojtech.adam@mendelu.cz
Received 30 October 2013; revised 25
November 2013; accepted 3 December
2013. Final version published online 30
December 2013.
MICROBIOLOGY LETTERS
DOI: 10.1111/1574-6968.12353
Editor: Jeff Cole
Keywords
antimicrobial effect; growth; inhibition;
nanotechnology.
Abstract
Interactions of silver phosphate nanoparticles (SPNPs) and selenium nanoparticles (SeNPs) with Staphylococcus aureus cultures have been studied at the cellular, molecular and protein level. Significant antibacterial effects of both SPNPs
and SeNPs on S. aureus were observed. At a concentration of 300 lM, SPNPs
caused 37.5% inhibition of bacterial growth and SeNPs totally inhibited bacterial growth. As these effects might have been performed due to the interactions
of nanoparticles with DNA and proteins, the interaction of SPNPs or SeNPs
with the amplified zntR gene was studied. The presence of nanoparticles
decreased the melting temperatures of the nanoparticle complexes with the
zntR gene by 23% for SeNPs and by 12% for SPNPs in comparison with the
control value. The concentration of bacterial metallothionein was 87% lower in
bacteria after application of SPNPs (6.3 lg mg 1 protein) but was increased by
29% after addition of SeNPs (63 lg mg 1 protein) compared with the
S. aureus control (49 lg mg 1 protein). Significant antimicrobial effects of the
nanoparticles on bacterial growth and DNA integrity provide a promising
approach to reducing the risk of bacterial infections that cannot be controlled
by the usual antibiotic treatments.
Introduction
Staphylococcus aureus is often a cause of infection in vascular grafts. Such infection may treated with appropriate
antibiotics, but most S. aureus strains have developed
resistance to available antibiotics, and resistance even to
new antibiotics is steadily growing (van Hal & Fowler,
2013). The use of metal-based drugs and nanoparticles is
one of the highly promising means to suppress infections
caused by antibiotic-resistant bacteria (Rai et al., 2009).
Synthesized silver and selenium nanoparticles (SeNPs)
have been found to be bactericidal and have been proved
to be good alternatives for the development of antimicrobial agents (Tran & Webster, 2011; Wang & Webster,
2013). Metallic nanoparticles interacting with cellular
components (DNA, RNA and ribosomes) deactivate and
FEMS Microbiol Lett 351 (2014) 195–201
effectively alter cellular processes (Grigor’eva et al., 2013).
It is assumed that silver nanoparticles can penetrate the
cell membrane to reach the cytosol due to their ability to
dissolve slowly while releasing Ag+ ions, but the exact
mechanism of the silver nanoparticles antimicrobial
action remains unclear.
Selenium has been studied for various medical applications and as a potential material for orthopaedic implants
(Wang & Webster, 2012). There also are studies indicating
an ability of selenium compounds to inhibit the growth of
bacteria and the formation of bacterial biofilms (Wang &
Webster, 2012). Among numerous selenium compounds,
2,4,6-tri-para-methoxyphenylselenopyrylium chloride, 9para-chlorophenyloctahydroselenoxanthene, and perhydroselenoxanthene have been demonstrated to have
antibacterial activity in vitro, especially against S. aureus.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
D. Chudobova et al.
196
However, the precise effect of elemental SeNPs remains
largely unknown (Tran & Webster, 2011; Ramos et al.,
2012).
In the past decade, study of the toxicological properties
of nanomaterials and/or nanoparticles has opened up a
new research field known as nanotoxicology (Yan et al.,
2013). Research on nanotoxicity is of extremely high scientific, social and economic value (Pumera, 2011). Nanomaterial-induced reactive oxygen species play a key role
in cellular and tissue toxicity (Yan et al., 2013). The toxicity of silver nanoparticles is enhanced in the presence of
H2O2, highlighting the biological relevance of investigating the oxidative dissolution of these nanoparticles (Ho
et al., 2010). In addition, it has been shown that SeNPs
can inhibit proliferation and induce apoptosis in promastigote forms of the protozoa Leishmania major (Beheshti
et al., 2013).
The aims of this study were to investigate the effects of
SeNPs and silver phosphate nanoparticles (SPNPs) on
inhibiting growth in S. aureus, morphologically altering
the bacteria cell structure and changing its biochemical
parameters, and thereby to identify a suitable substance
with antibacterial properties to reduce the risk of bacterial
(staphylococcal) infections. Another focus was to observe
effects of both SeNPs and SPNPs on bacterial metallothionein concentration and to study the interactions of these
nanoparticles with the amplified zntR gene.
Materials and methods
Cultivating S. aureus
Staphylococcus aureus (NCTC 8511) was obtained from
the Czech Collection of Microorganisms, Masaryk
University, CZ. Staphylococcus aureus was inoculated into
lysogeny broth medium for 24 h on a shaker at 40 g and
37 °C. The bacterial culture was diluted to
OD600 nm = 0.1 for all experiments. Petri dishes were
each covered with 3 mL of the S. aureus culture in lysogeny broth that had been grown for 24 h. Discs 1 cm in
diameter applied to Petri dishes with bacteria were cut
out from vascular grafts produced by the VUP Medical
(Czech Republic) and were mixed with SPNPs or SeNPs
in various concentrations. Dishes were incubated in a
growth chamber at 37 °C for 24 h. Growth was measured
by absorbance using a Multiskan EX apparatus (Thermo
Fisher Scientific, Germany). For measurement purposes,
S. aureus culture was mixed in the microplate with various concentrations of SPNPs and SeNPs. As a control,
the culture was also grown without nanoparticles. The
concentrations of SPNPs or SeNPs were 0, 10, 25, 50, 75,
150, 225 and 300 lM. Total volume in the microplate
wells was always 300 lL (Chudobova et al., 2013a, b).
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Analysing biochemical parameters of S. aureus
The effect of nanoparticles on S. aureus was tested using
STAPHYtest 24 biochemical detection tests (Erba Lachema,
Czech Republic) for the following substances: urease,
arginine, ornithine, b-galactosidase, b-glucuronidase,
b-glucosidase, phosphatase, esculin, N-acetyl-b-D-glucosamine, sucrose, mannitol, xylose, galactose, trehalose,
maltose, mannose, lactose, sorbitol, ribose, fructose, cellobiose, arabinose, xylitol and raffinose. Substances were
mixed with S. aureus bacterial culture with or without
added nanoparticles (300 lM). Complexes were measured
for 24 h using the Multiskan EX apparatus via ASCENT
Software for Multiskan.
Preparing and characterizing SPNPs and SeNPs
SPNPs were prepared by dissolving (di)sodium hydrogen
phosphate heptahydrate in water purified to the American
Chemical Society (ACS) standard and then adding a solution of SPNPs nitrate in ACS-standard water according to
Khan et al. (2012). The reaction proceeded immediately
with the formation of yellow colloidal nanoparticles
(200–300 nm in diameter; Chudobova et al., 2013a, b).
To the prepared SeNPs, sodium selenite pentahydrate
Na2SeO35H2O (26.3 mg) was dissolved in 50 mL ACSstandard water, and 3-mercaptopropionic acid (40 lL)
was then slowly added to the solution under stirring.
Afterwards, pH was adjusted to eight using 1 M NaOH.
The reaction mixture was stirred for 2 h. SeNPs were
stored in darkness at 4 °C. One millilitre of the solution
contains 158 lg of Se nanoparticles (50–100 nm in diameter). Chemicals used in this study were purchased from
Sigma-Aldrich in ACS purity unless noted otherwise.
SPNPs and SeNPs were measured on a Spectro Xepos
spectrometer (Spectro Analytical Instruments, Germany).
The samples were measured on a powder diffraction anode
X-ray tube at 47.63 kV and 0.5 mA current and were
detected with a Barkla scatter aluminium oxide. The sample was measured through the polyethylene bottle side wall
20 mm above the bottom. The SPECTRO XEPOS software and
TurboQuant method were applied for data analysis.
Cell microscopy
An Olympus IX 71 inverted system microscope (Japan)
was used for imaging of the cells. Total magnification was
16009. The parameters included an exposure time of
32 ms and ISO was set to 200. Structures were also characterized using an FEG-SEM MIRA XMU electron microscope (Tescan, Czech Republic). Samples were coated
with 10 nm of carbon using a K950X carbon coater
(Quorum Technologies, UK). For automated acquisition
FEMS Microbiol Lett 351 (2014) 195–201
197
Antimicrobial metal-based nanoparticles
of selected areas, the TESCAN
software tool was used.
IMAGE SNAPPER
proprietary
Determining total proteins
Pyrogallol red protein assay (Skalab, Czech Republic) is
based upon the formation of a blue protein-dye complex
in the presence of molybdate under acidic conditions
(pH 2.5). 150 lL of the reagent mixture (50 mM succinic
acid, 3.47 mM sodium benzoate, 0.06 mM sodium
molybdate, 1.05 mM sodium oxalate, and 0.07 mM pyrogallol red) was pipetted into a plastic cuvette. Then, 8 lL
of sample was added. Absorbance was measured at
k = 605 nm using a BS-400 chemistry analyser (Mindray,
China) after 6 min of incubation. The resulting value was
calculated from the absorbance values of the pure reagent
mixture and that of the sample after 6 min of incubation.
Differential pulse voltammetric determination
of metallothionein
Electrochemical measurements were performed on a 747
VA Stand instrument connected to a 746 VA Trace Analyzer and 695 Autosampler (Switzerland), using a standard cell with three electrodes and cooled sample holder
(4 °C). As a supporting electrolyte, Brdicka solution containing 1 mM Co(NH3)6Cl3 and 1 M ammonium buffer
[NH3(aq) and NH4Cl, pH 9.6] was used (Adam et al.,
2008).
Interaction of zntR gene from DNA with SPNPs
and SeNPs
The zntR gene was amplified from S. aureus according to
Singh et al. (1999). Spectra were recorded at 200–400 nm
after 120 min of interaction using 1-cm quartz cuvettes
(Hellma, UK) on a SPECORD 210 spectrophotometer
(Analytik Jena, Germany) at 25 °C. Denaturation of a
complex of amplified zntR gene with SPNPs and SeNPs
in eight concentrations (0, 10, 25, 50, 75, 150, 225 and
300 lM) was monitored spectrophotometrically using a
SPECORD S600 spectrophotometer with a diode detector
(Analytik Jena). The sample was incubated for 3 min at
increasing temperatures at 25–99 °C and the absorbance
was measured at 200–800 nm.
Determining protein mass spectra by matrixassisted laser desorption/ionization time of
flight (MALDI-TOF)
After cultivation overnight, 500 lL culture (0.1 = OD600 nm)
was centrifuged at 14 000 g for 2 min. Supernatant was
discarded and the pellet was suspended in 300 lL of
FEMS Microbiol Lett 351 (2014) 195–201
deionized water. Then, 900 lL of ethanol was added. After
centrifugation at 14 000 g for 2 min, the supernatant was
discarded and the obtained pellet was air-dried. The pellet
was then dissolved in 25 lL of 70% formic acid (v/v) and
25 lL of acetonitrile and mixed. The samples were
centrifuged at 14 000 g for 2 min and 1 lL of the clear
supernatant was spotted in duplicate onto an MTP 384 target (Bruker Daltonics) and air-dried at room temperature.
Each spot was overlaid with 1 lL of a-cyano-4-hydroxycinnamic acid matrix solution. Spectra were measured on a
Bruker MALDI-TOF/TOF apparatus in the m/z range of
2–20 kDa.
Results
Metal nanoparticles caused significant growth inhibition of
S. aureus, with application of SeNPs inducing twice as much
growth inhibition as SPNPs (7.0 0.5 and 3.0 0.5 mm,
respectively). There were also significant morphological
changes in the cell structure, these being most evident in the
nucleoid (Fig. 1A).
In addition, our synthesized nanoparticles (SPNPs or
SeNPs) were characterized with a scanning electron
microscope (Fig. 1B). Figure 1(B-d) shows the structure
of S. aureus culture without the addition of nanoparticles.
In the case of SPNPs, the formations were square or
spherical in character (Fig. 1B-e) and in the case of SeNPs only small spherical particles (Fig. 1B-f) were
observed, which confirmed our previous hypothesis.
After S. aureus had been exposed for 18 h to SPNPs,
inhibition of bacterial growth was observed and absorbance
values at 600 nm had decreased from 0.4 to 0.25 a.u.
(Fig. 2A-a). In applying the SPNP concentration with the
weakest antibacterial activity (10 lM) to the bacterial culture, absorbance values comparable to those of the control
group (0.4) were obtained. The greatest decrease in absorbance values (by 38%) in comparison with the control was
achieved by applying 300 lM SPNPs (Fig. 2A-a). By contrast, in the case of SeNPs, the minimum inhibitory concentration was achieved when 10 lM SeNPs was applied
and the total inhibitory concentration occurred when
300 lM SeNPs was applied (Fig. 2A-b). Half maximal
inhibitory concentration values confirmed the obtained
results, as IC50 was 268.2 for SPNPs and 4.4 lM for SeNPs.
We also investigated changes in the biochemical
parameters of S. aureus after the addition of nanoparticles. Twenty-four hours after application of SPNPs
(300 lM), changes in 11 biochemical tests (of 24 tests in
total) were observed (Fig. 2B-b). After application of
SeNPs, the results of only seven tests were different from
those of the control group (Fig. 2B-c).
Because we observed changes in the morphology of the
bacterial cell nucleoid after exposure of S. aureus to
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
D. Chudobova et al.
198
(A) a
b
c
3 mm
0 mm
cell wall
nucleoid
(B) d
7 mm
nucleoid
e
f
Absorbance (AU)
a
3.0
2.0
1000
AgKα
f
b
500
SPNPs
AgKβ
0
800
1000
Intensity (counts)
Intensity (counts)
(C) 4.0
10 000
Channel (keV)
c
SeKα
5000
SeKβ
0
440
490
540
Channel (keV)
1.0
SeNPs
0.0
230
330
430
530
630
Wavelength (nm)
Absorbance (AU)
Absorbance (AU)
(A)
Fig. 1. (A) Growth inhibition zones in
bacterial culture after application of 300 lM
of nanoparticles and a microscopic view of
cells: (a) Staphylococcus aureus, (b) after
application of SPNPs, (c) after application of
SeNPs. (B) Bacterial culture and nanoparticles
under electron microscopy: (d) S. aureus, (e)
SPNPs, (f) SeNPs. Images are in different
scales, with 1 unit of the scale corresponding
to 5 lm in B-d, 500 nm in B-e, and 1 lm in
B-f. (C) Absorption spectra of 300 lM of
SPNPs and SeNPs: (a) visualization in ambient
light: MilliQ water, SPNPs, SeNPs; X-ray view
of content elements: (b) SPNPs; (c) SeNPs.
(B)
0.5
a
IC50 = 268.2 μM
0.4
a
b
0.3
0.2
0.1
0.0
0
6
12
18
24
Time (h)
0.4
b
IC50 = 4.4 μM
0.3
0.2
0.1
0.0
0
6
12
18
24
Identical compared to control S. aureus
Different compared to control S. aureus
Time (h)
SPNPs or SeNPs, we investigated the interaction of both
nanoparticle types with DNA using amplified zntR gene.
After application of the various concentrations of SeNPs
(0, 10, 25, 50, 75, 150, 225 and 300 lM) there was a
marked increase in absorbance signal at 260 nm with
increasing concentrations of SeNPs (Fig. 3A-b) and composition of nanoparticles, but this shift was not observed
when SPNPs were applied (Fig. 3A-a). Statistically significant changes were observed in the melting temperatures
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Fig. 2. (A) Spectrophotometric analysis of the
growth of S. aureus bacterial culture with (a)
SPNPs and (b) SeNPs in concentrations 0, 10,
25, 50, 75, 150, 225 and 300 lM.
(B) Biochemical parameters of (a) S. aureus, (b)
S. aureus with 300 lM SPNPs, and (c)
S. aureus with 300 lM SeNPs.
of the amplified zntR gene in complexes with SPNPs or
SeNPs at the highest concentration of 300 lM (Fig. 3B).
Differential changes in the melting temperature of amplified zntR gene probably can be caused by different affinity
of nanoparticles to the bacterial DNA. A greater decrease
in melting temperature in comparison with the control
was observed in the interaction of SPNPs and amplified
zntR gene, than in the interaction of SeNPs with amplified zntR gene (23% vs. 12%, respectively). SPNPs may
FEMS Microbiol Lett 351 (2014) 195–201
199
Antimicrobial metal-based nanoparticles
0.08
0.12
0.08
0.04
0.00
0
150
300
Concentration of
SPNPs (μM)
200
75
300
150
0.20
0.16
0.04
0.00
50
400
500
b
0.16
0.12
0.08
0.04
0.00
600
Amount of MT
(μg mg–1 of protein)
60
0
150
200
300
400
*
*
300
500
Wavelength (nm)
70
(B)
300
0.20
0.16
0.12
0.08
0.04
0.00
Concentration of
SeNPs (μM)
Wavelength (nm)
(C)
225
600
Temperature melting
point (°C)
0.12
25
Absorbance (AU)
0.16
10
0.20
Absorbance (AU)
Absorbance (AU)
a
Absorbance (AU)
0
(A) 0.20
100
*
80
*
60
40
20
0
S. aureus
SPNPs
SeNPs
Control strainor S. aureus
with nanoparticles
(D)
a
50
40
b
30
20
10
0
c
S. aureus
SPNPs
SeNPs
Control strain or S. aureus
with nanoparticles
Fig. 3. (A) Spectrophotometric determination of amplified zntR gene after application of different concentrations (0, 10, 25, 50, 75, 150, 225
and 300 lM) of nanoparticles: (a) SPNPs, (b) SeNPs. (B) Changes of bacterial DNA melting temperature after application of SPNPs and SeNPs. (C)
Determination of metallothionein in control strain and after application of silver and SeNPs. (B and C) Data represent the mean SD from three
measurements, *P < 0.05. (D) MALDI-TOF mass spectra protein fingerprints for the identification of (a) Staphylococcus aureus and S. aureus after
application of (b) SPNPs or (c) SeNPs.
have a greater affinity for the amplified zntR gene than
SeNPs.
The concentration of bacterial metallothionein was significantly lower (by about 87%) in bacterial cells after
application of SPNPs (6.3 lg mg 1 protein) and a higher
(about 29%) concentration of metallothionein occurred in
bacterial cells after addition of SeNPs (63 lg mg 1 protein) as compared with the S. aureus control (49 lg mg 1
protein; Fig. 3C). The presence of the zntR gene, which
encodes metallothionein formation, was confirmed by
polymerase chain reaction (results not shown). The effect
of nanoparticles probably caused a change in the protein
composition of the S. aureus culture, as was demonstrated
by MALDI-TOF mass spectrometry, although the change
was not statistically significant (Fig. 3D).
Discussion
Advances in nanotechnology, and particularly in the preparation of metal nanoparticles, can be considered to constitute one of the keys to developing new vascular grafts,
because silver ions can lead to the prevention of bacterial
infection (Ho et al., 2013). In addition, SeNPs have been
found to have high antibacterial activity and they can be
FEMS Microbiol Lett 351 (2014) 195–201
easily prepared (Ramos et al., 2012). The antibacterial
activity of SeNPs was greater than that of the SPNPs used
in this study, and it was comparable to that of the smaller
SPNPs (18–21 nm) used by Prabakar et al. (2013). This
probably was because we prepared SeNPs that were smaller
in size (50–100 nm) than the SPNPs (200–300 nm). The
present study showed that the silver nanoparticles exhibited
antibacterial activity even at nanoparticle sizes equal to
hundreds of nm. When comparing our findings on inhibition of S. aureus growth in the presence of selenium to
those from other scientists, we found that our results were
similar to those of Naik et al. (2009). However, our
colonies were smaller than those of Naik et al., probably
because they had used selenium in a complex with
2-selenobenzo[h]quinoline-3-carbaldehyde.
The effect of 40–60 nm SeNPs on S. aureus was studied
by Tran & Webster (2011). In that work, bacterial growth
was inhibited by SeNPs in the concentration range 7.8–
31 lg mL 1 to approximately 1.7% of that seen in the
controls and the SeNPs killed approximately 40% of
S. aureus cells. Antimicrobial activity of SeNPs was also
confirmed in the work of Hariharan et al. (2012).
In our study the highest used concentration of SeNPs
(300 lM, 23.7 lg mL 1) caused total growth inhibition
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
D. Chudobova et al.
200
within 24 h. Similar results for a 24-h inhibition period in
relation to S. aureus growth on selenium nanoparticlecoated polyvinyl chloride are presented in the work of
Ramos et al. (2012). The most interesting finding of our
study is that the total inhibition concentration of SeNPs
should be at least 5 lg mL 1, which corresponds with
results published by Tran & Webster (2011). Biochemical
parameters showed greater similarity between S. aureus
with the addition of SeNPs and the control, but greater
differences with the addition of SPNPs.
Our study also demonstrated the interaction of DNA
with SeNPs, whereby these particles probably impair the
DNA structure of the zntR gene amplified in vitro. This
statement is supported by the study of Beheshti et al.
(2013), which reported increasing concentrations of
SeNPs biosynthesized by Bacillus sp. MSH-1-induced
cytotoxic effects and apoptosis in promastigote forms of
the protozoa L. major. Apoptosis was demonstrated by
DNA fragmentation in the concentration range 1–
150 lg mL 1 of SeNPs. A similar toxicity effect of SeNPs
on genomic DNA had been reported by Chen et al.
(2008) for human melanoma cells treated with chemically
synthesized SeNPs. Treatment of A375 human melanoma
cells with SeNPs resulted in dose-dependent cell apoptosis, as indicated by DNA fragmentation and phosphatidylserine translocation (Chen et al., 2008).
In our study, the results regarding concentration of metallothionein were comparable to those of Dar et al. (2013).
Their group was interested in the production of metallothionein in various microorganisms in response to cadmium
and they showed an increased accumulation of cadmium in
cells expressing the protein metallothionein in comparison
with control cells. Our results show a distinct decrease in
metallothionein synthesis after addition of SPNPs to
S. aureus. In contrast, only a slight increase was observed
after the addition of SeNPs. This may be associated with a
similarity in the biochemical parameters of S. aureus with
and without the addition of SeNPs. After the addition of
SPNPs, S. aureus showed more differences in biochemical
parameters. After application of both SPNPs and SeNPs, on
the other hand, S. aureus bacterial culture showed only
minor differences in MALDI-TOF mass spectra protein fingerprints in comparison with the control. This leads to the
conclusion that smaller nanoparticles (SeNPs) have a
greater inhibitory effect compared with larger nanoparticles
(SPNPs). Our results suggest an effective way to prevent
S. aureus infections using nanostructured selenium.
Acknowledgements
Financial support from Sensor, Information and Communication Systems CZ.1.05/2.1.00/03.0072 is gratefully
acknowledged. The authors declare no conflict of interest.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
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